I. Field of the Invention
The invention relates generally to antibody selection and immunoassay technology and specifically to remarkably efficient and rapid polypeptide library screening methods. The invention also includes competitive immunoassays for analyte detection. The disclosed methods open the way for production of kilogram quantities of antibodies from inexpensively maintained microorganism cultures.
II. Description of Related Art
Antibodies are of increasing importance in human therapy, assay procedures and diagnostic methods. However, methods of identifying antibodies and production of antibodies is often expensive, particularly where monoclonal antibodies are required. Hybridoma technology has traditionally been employed to produce monoclonal antibodies, but these methods are time-consuming and result in isolation and production of limited numbers of specific antibodies. Additionally, relatively small amounts of antibody are produced; consequently, hybridoma methods have not been developed for a large number of antibodies. This is unfortunate as the potential repertoire of immunoglobulins produced in an immunized animal is quite high, on the order of &gt;10.sup.10, yet hybridoma technology is too complicated and time consuming to adequately screen and develop large number of useful antibodies.
One approach to this problem has been the development of library screening methods for the isolation of antibodies (Huse et al, 1989; McCafferty et al, 1990; Chiswell & McCafferty, 1992; Chiswell & Clackson, 1992; Clackson, 1991). Functional antibody fragments have been produced in E. coli cells (Skerra & Pluckthun, 1988; Better et al, 1988; Orlandi et al, 1989; Sastry et al, 1989) as "libraries" of recombinant immunoglobulins containing both heavy and light variable domains (Huse et al, 1989). The expressed proteins have antigen-binding affinity comparable to the corresponding natural antibodies. However, it is difficult to isolate high binding populations of antibodies from such libraries and where bacterial cells are used to express specific antibodies, isolation and purification procedures are usually complex and time-consuming.
Combinatorial antibody libraries generated from phage lambda (Huse et al, 1989) typically include millions of genes of different antibodies but require complex procedures to screen the library for a selected clone. Methods have been reported for the production of human antibodies using the combinatorial library approach in filamentous bacteriophage. A major disadvantage of such methods is the need to rely on initial isolation of the antibody DNA from peripheral human blood to prepare the library. Moreover, the generation of human antibodies to toxic compounds is not feasible owing to risks involved in immunizing a human with these compounds.
Currently the most widely used approach for screening polypeptide libraries is to display polypeptides on the surface of filamentous bacteriophage (Smith, 1991; Smith, 1992). The polypeptides are expressed as fusions to the N-terminus of a coat protein. As the phage assembles, the fusion proteins are incorporated in the viral coat so that the polypeptides become displayed on the bacteriophage surface. Each polypeptide produced is displayed on the surface of one or more of the bacteriophage particles and subsequently tested for specific ligand interactions. While this approach appears attractive, there are numerous problems, including difficulties of enriching positive clones from phage libraries. Enrichment procedures are based on selective binding and elution onto a solid surface such as an immobilized receptor. Unfortunately, avidity effects arise due to multivalent binding of the phage and the general tendency of phage to contain two or more copies of the displayed polypeptide. The binding to the receptor surface therefore does not depend solely on the strength of interaction between the receptor and the displayed polypeptide. This causes difficulties in the identification of clones with high affinity for the receptor; thus, there remain distinct deficiencies in the methods used to isolate and screen polypeptides, particularly antibodies, even in view of the development of phage libraries.
Moreover, there is a significant need for procedures that are rapid and inexpensive relative to cost of currently used techniques.
Immunoassays have typically been used for the detection of antigens or antibodies, less frequently for determination of other classes of compounds. Immunoassays can be generally divided into two types: heterogeneous assays requiring multiple separation steps, and homogeneous assays which are performed directly. Heterogeneous immunoassays in general involve a ligand or antibody immobilized on a solid matrix. A sample containing an analyte is contacted with the immobilized antibody and the amount of complex formed on the matrix support is determined from a label attached directly or indirectly to the immobilized complex.
Heterogeneous immunoassays may be performed as sandwich assays in which a molecule of interest is reacted with an immobilized antibody that specifically binds that molecule with high affinity. In a second step, a conjugate formed from the same or different antibody to the antigen and a marker molecule is reacted with the antigen-antibody complex on the immobilization matrix. After removal of excess free marker conjugate, the bound marker conjugate, which is proportional to the amount of analyte in the sample, is measured.
ELISA or enzyme-linked immunosorbent assay is one example of an immunoassay. ELISAs are extensively used in biotechnology applications, particularly as immunoassays for a wide range of antigenic substances. The sensitivity of ELISA is based on the enzymatic amplification of the signal.
However, despite such widespread use, ELISA has several disadvantages, including inconvenience and expense that preclude more general use. Additionally, immunoassays depend on the availability of antibodies, which is frequently a major consideration in terms of cost or feasibility of a specific assay. Several washing steps may often be required in ELISA procedures to remove excess antibody not bound to the immobilization support or to remove excess analyte or antibody following the primary binding reaction. The steps necessary to remove excess enzyme-conjugate that does not bind to the immobilized antibody-analyte complex may significantly increase assay time and cost.
There has been some effort to provide semi-automation of ELISA, for example the apparatus and method described in U.S. Pat. No. 4,981,785. However, these methods involve not only the expense of the equipment and training of specialized personnel, but also fail to provide rapid and economical quantitation of analytes.
Unfortunately, alternative analytical methods for quantitative determination of trace amounts of many analytes, including not only polypeptides but also organic species such as toxic chemicals, typically involve relatively complex and inconvenient procedures. In most cases, samples must be collected, extracted and prepared for specific analyses. Procedures frequently involve use of hazardous chemicals such as organic solvents and/or radioactive material, thereby adding to cost.
Recent advances in biotechnology, specifically the advent of monoclonal antibodies and the use of enzyme labels in place of radiolabels, have promoted a rapid expansion of immunoassay applications to include the detection of certain toxic chemicals (Collins, 1985; Gould and Marx, 1988; Stanker, 1989; Watkins, 1989; Roberts, 1989). Enzyme labels, for example, are particularly useful because the catalytic properties of the enzyme provide powerful biochemical amplification, thereby allowing detection of extremely low analyte concentrations. However, the utility of this type of detection may be limited if the binding site of the antibody is not available or the sample solution contains interfering substances.
Enzyme-amplified immunoassay techniques have been applied to the determination of different drug species. In this system a hapten-enzyme conjugate is prepared so that enzyme activity is retained after conjugation. When the conjugate binds with the hapten-specific antibody there is a loss in enzyme activity. Any free hapten (drug) in a sample reduces the inhibition by competing for antibody binding sites. Enzyme activity is thus proportional to concentration of free hapten. This type of assay has been developed by the Syva Company (Palo Alto, Calif.) under the trademark EMIT. However, the EMIT assay is less satisfactory for detection of relatively large molecules such as proteins.
Immunodetection technology employing solid phase enzyme immunoassay for detection of certain environmental waste by-products has been reported (Huber, 1985). For example, the agricultural chemical Atrazine was reported detectable in the 1.1 to 2200 ppb range. Enzyme immunoassay (EIA) sensitivity was further improved by using spheres as antibody carriers and by using affinity purified antibodies. While this is a step forward in the ability to detect low levels of environmental contaminants, the procedures are time-consuming and costly.
Immunoassays represent a powerful technique for the identification and quantification of specific molecules. Radio Immunoassay (RIA) and ELISA techniques currently form the basis of many medical diagnostic procedures. However, solid phase immunoassays rely on isolated and purified antibodies that may be prohibitively expensive. In cases where the detection reagent is an enzyme or radiolabeled material, additional considerations are safety, equipment and technical manpower cost.
Despite some progress and ongoing efforts to expand the availability and identification of useful antibodies for immunoassays, there is a lack of rapid and inexpensive antibody screening methods. Available technologies have failed to provide means to cost effectively produce large quantities of such antibodies. Additionally, despite the wide application of immunoassay methods, most are complex, often slow and frequently employ hazardous reagents.